Macro CD5L+ deteriorates CD8+T cells exhaustion and impairs combination of Gemcitabine-Oxaliplatin-Lenvatinib-anti-PD1 therapy in intrahepatic cholangiocarcinoma

Intratumoral immune status influences tumor therapeutic response, but it remains largely unclear how the status determines therapies for patients with intrahepatic cholangiocarcinoma. Here, we examine the single-cell transcriptional and TCR profiles of 18 tumor tissues pre- and post- therapy of gemcitabine plus oxaliplatin, in combination with lenvatinib and anti-PD1 antibody for intrahepatic cholangiocarcinoma. We find that high CD8 GZMB+ and CD8 proliferating proportions and a low Macro CD5L+ proportion predict good response to the therapy. In patients with a poor response, the CD8 GZMB+ and CD8 proliferating proportions are increased, but the CD8 GZMK+ proportion is decreased after the therapy. Transition of CD8 proliferating and CD8 GZMB+ to CD8 GZMK+ facilitates good response to the therapy, while Macro CD5L+–CD8 GZMB+ crosstalk impairs the response by increasing CTLA4 in CD8 GZMB+. Anti-CTLA4 antibody reverses resistance of the therapy in intrahepatic cholangiocarcinoma. Our data provide a resource for predicting response of the combination therapy and highlight the importance of CD8+T-cell status conversion and exhaustion induced by Macro CD5L+ in influencing the response, suggesting future avenues for cancer treatment optimization.

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Reporting for specific materials, systems and methods We did not perform analysis based on sex and gender in our study.Both females and males were enrolled in our study without any prejudice.
No particular attention given to race, ethnicity, or other socially pertinent factors.
The characteristics of the enrolled patients were listed in Supplementary Data1.
A total of 55 patients with GOLP treatment in the two cohorts were enrolled in this study (27 patients in NCT03951597, 28 patients in FDU-ZS-iCCA-T).Another 591 patients in the two GOLP treatment-naive cohorts,including 262 patients in FU-iCCA (Cancer Cell. 2022;40:70-87.e15) and 329 patients in FDU-ZS-iCCA were also enrolled into this study.All the patients were diagnosed with iCCA pathologically in Zhongshan Hospital, Fudan University.Detailed clinical information of the patients was showed in Supplementary Data1.
Written informed consent for clinical information collection and tissue collection was obtained from all the patients, and the collection of human samples and clinical information was approved by the Zhongshan Hospital Research Ethics Committee Raw sequencing data of scRNA and TCR generated in this study have been deposited in the National Genomics Data Center with the accession co de (PRJCA021882) [https://ngdc.cncb.ac.cn/bioproject/browse/PRJCA021882], with a copy deposition in biosino NODE database (OEP003206) [htt ps://www.biosino.org/node/project/detail/OEP003206].Data are available upon request through the repository portal.The raw sequencing data is onl y available for non-commercial and academic purposes under controlled access due to ethical and legal restrictions.The corresponding authors will respond to requests for the data in two weeks.The data will be available for three months once access has been granted.The publicly available dat a of bulkRNA and clinicopathologic information used in this study are available in the Supplemental information ( No data was excluded.
All samples represent a minimum of three replicates and the attempts at replication were successful.
Animals in this experiment were randomly assigned to different groups before treatment.
Blinding was not performed during animal treatment in this study since lenvatinib showed obviously different color to other regimens.The investigators were blinded to group allocation during data collection and analysis.

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A numerical value for number of of cells or or percentage (with statistics) is is provided.Positive and negative boundaries were determined by by the unstain control (see Figure S5F)

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to to confirm that a figure exemplifying the gating strategy is is provided in in the Supplementary Information.This study does not involve plants.This study does not involve plants.This study does not involve plants.Isolated single-cell suspensions from the tissue were centrifuged (350Xg, 6min, 4°C) and resuspended in in 100 l Cell Staining Buffer (Biolegend, Cat.420201).Human TruStain FcX™ (Fc Receptor Blocking Solution, BioLegend Cat.No. 422301) was used to to incubate with the single-cell suspensions for 15 15 min at at room temperature before cell-surface staining.Beckman MoFlo XDP; BD BD LSRFortessa™ X-20 Flow Cytometer FlowJo 10.6.2 30000 cells were sorted per sample.Positive and negative boundaries were determined by by the unstain control.
Table S1) of the paper [https://www.cell.com/cancer-cell/fulltext/S1535-6108(21)00659-0#supplementaryMaterial]69.The cisTarget Human motif database used by SCENIC method88 i n this study are available in https://resources.aertslab.org/cistarget/motif2tf/motifs-v9-nr.flybase-m0.001-o0.0.tbl.The remaining data are available wi thin the Article, Supplementary Information or Source Data file.Source data are provided with this paper.The sample size is showed in the Figure Legends or in the Figures.The sample sizes employed in this study are consistent with previously published papers in Nature communications (Nat Commun 14, 8175 (2023).Nat Commun 14, 7863 (2023))